Understanding Neural Development--Use of Mouse Mutations

癫痫模型与钙通道功能 P/Q型电压依赖性钙通道CACNA 1A突变在人类中引起显性遗传性偏头痛、发作性共济失调和小脑萎缩，在小鼠中引起复发性遗传性共济失调、发作性运动障碍、小脑萎缩和失神癫痫。这些物种差异的基础和疾病机制尚不清楚。为了解决这个问题，并确定所需的P/Q功能在体内，我们创建了一个种系Cacna 1a无效突变（指定Cacna 1aFcrtm 1）的基因打靶。小白鼠以一种特殊的模式发展成肌张力障碍和迟发性小脑变性。这表明在小脑神经元的一个子集的生存需要P/Q功能。纯合无效小鼠完全缺乏P/Q型通道活性，并且它们也缺乏w-CTx-MVIC受体，表明单个基因编码P/Q通道活性。在P/Q无效颗粒细胞中检测到L-和N-型电流密度的增加。杂合子Cacna 1aFcrtm 1/+小鼠表型正常，尽管电流密度降低50%，表明电流密度降低本身不足以引起共济失调和癫痫发作的自发性小鼠突变体的病理生理学。 据推测，R型钙电流的结果从Cacna 1 e基因的表达。为了验证这一假设，我们研究了小鼠中Cacna 1 e通道亚基被删除的电压依赖性钙通道的特性。应用ω-芋螺毒素GVIA，ω-蝮蛇毒IVA，尼莫地平从野生型小鼠培养的小脑颗粒神经元抑制组件的全细胞Ba电流，留下一个“残余”的R电流的振幅约为30%的总Ba电流。该R电流的一小部分被Cacna 1 e选择性毒素SNX-482抑制，表明其由Cacna 1 e的表达引起。然而，SNX-482不抑制大部分R电流。SNX-482敏感部分的颗粒细胞R电流Cacna 1 e基因敲除小鼠缺席。我们还确定了一个亚群的背根神经节（DRG）神经元从野生型小鼠表达SNX-482敏感的R电流的组成部分。然而，与颗粒细胞一样，大多数DRG R电流不被SNX-482阻断。我们从这些实验中得出结论，存在一个组件的R电流，结果从Cacna 1 e钙通道亚基的表达，但大部分的R电流必须从其他钙通道α亚基的表达。 耳聋模型 waltzer（v）基因座的突变会导致内耳神经上皮细胞变性导致耳聋和前庭功能障碍。我们发现，waltzer编码一种新的钙粘蛋白（Cdh 23），这是最密切相关的果蝇脂肪蛋白。预测vJ等位基因中的单核苷酸缺失和v等位基因中的单核苷酸插入会截短N末端附近的每个蛋白质并产生功能性无效等位基因。原位杂交分析表明，Cdh 23在内耳的感觉毛细胞中表达，在那里它被认为是静纤毛交联的关键分子。此外，Cdh 23在椭圆囊囊孔、汇合管和Reissner膜中表达，表明Cdh 23也可能参与维持内淋巴的离子组成。最后，人类CDH 23中的突变最近被描述为两个基因座，DFNB 12和USH 1D，它们导致非综合征性耳聋，将Waltzer确定为人类听力损失的小鼠模型。 神经退化模型 对于共济失调（axJ）突变纯合子的小鼠在2-3周龄时发生严重震颤，随后在6周龄时发生后肢麻痹和死亡。最近，我们已经证明axJ编码泛素特异性蛋白酶14（Usp 14）。USP包含特异性切割泛素缀合物的半胱氨酸蛋白酶的大家族。虽然Usp 14可以切割泛素标记的蛋白质，但它不能加工多聚泛素，而多聚泛素被认为与帕金森病（PD）、脊髓小脑共济失调1型（SCA 1）和薄轴突营养不良（GAD）中的蛋白质聚集体有关。因此，Usp 14的生理底物可能含有短的泛素侧链，其去除可以调节诸如蛋白定位和蛋白活性的过程。由于在Usp 14的内含子5中插入了脑池内A颗粒（IAP），AxJ小鼠中Usp 14的表达显著改变。原位杂交研究表明，Usp 14在成人中枢神经系统（CNS）内的各种神经元中表达，包括大脑皮层、海马、浦肯野细胞和小脑深核。然而，与人类的PD和SCA 1或小鼠的GAD等神经退行性疾病不同，axJ CNS中没有可检测到的泛素化蛋白聚集体，也没有神经元细胞损失。相反，我们确定在axJ中枢神经系统的突触连接缺陷，这表明，USP可能是重要的因素，在调节突触的活动，可能通过神经递质的释放。

Epilepsy Models and Calcium Channel Function P/Q-type voltage-dependent calcium channel CACNA1A mutations cause dominantly inherited migraine, episodic ataxia, and cerebellar atrophy in humans and cause recessively inherited ataxia, episodic dyskinesia, cerebellar atrophy, and absence epilepsy in mice. The basis of these species differences and the disease mechanism(s) are not understood. To address this question and to identify required P/Q function in vivo, we created a germline Cacna1a null mutation (designated Cacna1aFcrtm1) by gene targeting. Null mice develop dystonia and late-onset cerebellar degeneration in a specific pattern. This indicates a requirement for P/Q function for survival in a subset of cerebellar neurons. Homozygous null mice completely lack P/Q-type channel activity, and they also lack w-CTx-MVIIC receptors, indicating that a single gene encodes P/Q channel activity. An increase of L- and N-type current densities is detected in P/Q-null granule cells. Heterozygous Cacna1aFcrtm1/+ mice are phenotypically normal, despite having a 50% reduction in current density, indicating that reduced current density is not itself sufficient to cause the pathophysiology of spontaneous mouse mutants with ataxia and seizures. It has been hypothesized that R-type Ca currents result from the expression of the Cacna1e gene. To test this hypothesis, we examined the properties of voltage-dependent Ca channels in mice in which the Cacna1e channel subunit had been deleted. Application of omega-conotoxin GVIA, omega-agatoxin IVA, and nimodipine to cultured cerebellar granule neurons from wild-type mice inhibited components of the whole-cell Ba current, leaving a "residual" R current with an amplitude of approximately 30% of the total Ba current. A minor portion of this R current was inhibited by the Cacna1e-selective toxin SNX-482, indicating that it resulted from the expression of Cacna1e. However, the majority of the R current was not inhibited by SNX-482. The SNX-482-sensitive portion of the granule cell R current was absent from Cacna1e knockout mice. We also identified a subpopulation of dorsal root ganglion (DRG) neurons from wild-type mice that expressed an SNX-482-sensitive component of the R current. However, as with granule cells, most of the DRG R current was not blocked by SNX-482. We conclude from these experiments that there exists a component of the R current that results from the expression of the Cacna1e Ca channel subunit but that the majority of R currents must result from the expression of other Ca channel alpha subunits. Deafness Models Mutations at the waltzer (v) locus result in deafness and vestibular dysfunction due to degeneration of the neuroepithelium within the inner ear. We showed that waltzer encodes a novel cadherin (Cdh23), which is most closely related to the Drosophila fat protein. A single nucleotide deletion in the vJ allele and a single nucleotide insertion in the v allele are predicted to truncate each protein near the N-terminus and produce a functional null allele. In situ hybridization analysis showed that Cdh23 is expressed in the sensory hair cells of the inner ear, where it has been suggested to be a molecule critical for crosslinking of the stereocilia. In addition, Cdh23 is expressed in the utriculo-saccular foramen, the ductus reuniens, and Reissner's membrane, suggesting that Cdh23 may also be involved in maintaining the ionic composition of the endolymph. Finally, mutations in human CDH23 have recently been described for two loci, DFNB12 and USH1D, which cause nonsyndromic deafness, identifying waltzer as a mouse model for human hearing loss. Neural Degeneration Models Mice homozygous for the ataxia (axJ) mutation develop severe tremors by 2-3 weeks of age, followed by hind limb paralysis and death by 6 weeks of age. Recently, we have shown that axJ encodes ubiquitin-specific protease 14 (Usp14). USPs comprise a large family of cysteine proteases that specifically cleave ubiquitin conjugates. Although Usp14 can cleave a ubiquitin-tagged protein, it is unable to process polyubiquitin, which is believed to be associated with the protein aggregates in Parkinson's disease (PD), spinocerebellar ataxia type 1 (SCA1), and gracile axonal dystrophy (GAD). The physiological substrate of Usp14 may therefore contain a short ubiquitin side chain, the removal of which may regulate processes such as protein localization and protein activity. Expression of Usp14 is significantly altered in axJ mice due to the insertion of an intracisternal-A particle (IAP) into intron 5 of Usp14. In situ hybridization studies show that Usp14 is expressed in a variety of neurons within the adult central nervous system (CNS), including the cerebral cortex, hippocampus, Purkinje cells, and deep cerebellar nuclei. However, unlike neurodegenerative disorders such as PD and SCA1 in humans or GAD in mice, there are no detectable ubiquitinated-protein aggregates nor is there neuronal cell loss in the axJ CNS. Instead, we identify synaptic connection defects in the axJ CNS, suggesting that USPs may be important factors in regulating the activity of synapses, possibly through neurotransmitter release.